Interpretive Summary: Extending the storage life of fruits and vegetables in order to supply them to consumers on more than a seasonal basis is done routinely in the produce industry. However, pathological, texture and flavor quality become limiting factors affecting consumer acceptance. Apples are stored for up to a year sometimes before marketing. The peel tissue of apples produces several volatile compounds called terpenes that contribute to the aroma of ripening fruit. Specific terpenes play a role in either the attraction of or defense against mite and insect pests, as well as development of a costly storage disorder known as superficial scald. We are characterizing the genes and enzymes involved in apple terpene synthesis in an effort to understand how their production is increased by cues such as ripening, wounding, feeding insects, and low temperature. In this study, a gene from apple peel was isolated that codes for an enzyme that performs the last step in synthesis of the terpene nerolidol. This compound is converted to a potent attractant of female codling moths seeking to lay their eggs. It was found that the nerolidol synthase gene is strongly activated by ethylene, the gaseous plant hormone that is released during ripening and after wounding. A long-range goal of this work is to limit attractant production in apple fruit by molecular genetic means, and thereby lower the incidence of codling moth infestation. This outcome will benefit both the apple industry and consumers by reducing the number of unsalable insect-damaged fruit and the need to frequently apply pesticides to maintain quality during storage.

Technical Abstract:
Increased production of terpenes and many other aroma-related volatiles occurs with the onset of ripening in apple fruit. The gaseous plant hormone ethylene plays a key role in the induction of volatile synthesis, but the mechanism is not yet understood. Using a degenerate primer based on a short conserved sequence shared by several sesquiterpene synthases, RT-PCR with RNA isolated from peel tissue of 'Law Rome' apples yielded an ~800-bp gene fragment. This was used to screen a cDNA library generated from the peel tissue mRNA. A full-length terpene synthase (TS) cDNA 1808 nucleotides long was isolated. The 1605-bp open reading frame encodes a protein 535 amino acids long with a molecular mass of 63 kDa. Sequence analysis of the apple TS showed it to be most similar to several linalool synthases. Oddly, the TS includes an RR(N8)W motif near the N-terminus but lacks the plastid transit peptide sequence common to monoterpene synthases. Expression of the apple TS gene in E. coli gave myc-epitope-tagged and untagged proteins estimated at ~67 and ~65 kDa, respectively. After urea extraction from bacterial inclusion bodies and renaturation, both proteins yielded one major product in TS assays with farnesyl diphosphate as substrate. GC-MS analysis showed the retention time and EI mass spectrum of the product to be identical to those of an(E)-nerolidol standard. Similar assays with geranyl diphosphate as substrate and conditions modified for monoterpene synthases failed to yield any monoterpene product. This is the first report of a nerolidol synthase gene, ANS1. RNA gel blots showed that ANS1 transcript increased over twofold in apple peel tissue during the first 4 weeks of fruit storage at 0.5 C. In contrast, ANS1 mRNA declined to nearly undetectable levels after 8 weeks of storage in fruit treated at harvest with a blocker of ethylene action, 1-methylcyclopropene.